The Secretory Mechanisms and Dysfunction Section investigates the molecular nature and function of the ion transport mechanisms involved in the fluid and electrolyte secretion process in the exocrine salivary gland. We are probing the structure-function relationships of cotransporter, exchanger and channel proteins using a combination of molecular biology, gene modification, proteomics and functional studies in mouse and human salivary glands. Accomplishments/conclusions: Calcium activated Cl- channel: Activation of an apical calcium activated Cl- channel (CaCC) triggers the secretion of saliva. To test the role of Tmem16A channels in the adult salivary gland we generated a conditional knockout mouse lacking Tmem16A expression in acinar cells (Tmem16A-/-). Ca2+-dependent salivation was abolished in Tmem16A-/- mice, confirming that the Tmem16A channel is obligatory for Ca2+-mediated fluid secretion. Beta-adrenergic, cAMP-induced salivation: The beta-adrenergic, cAMP-induced salivation mechanism is unclear. Does cAMP directly activate secretin or does the increase in cAMP activate a calcium-dependent process? We asked whether Tmem16A might be involved in beta-adrenergic, cAMP-induced salivation. Surprisingly, the amount of saliva secreted by Tmem16A-/- mice in response to thebeta-adrenergic receptor agonist isoproterenol (IPR) was comparable to littermate controls. Cftr and ClC-2 are not involved in fluid secretion: If beta-adrenergic, cAMP-induced salivation does not involve an increase in calcium and activation of Tmem16A, then what is the mechanism? Cftr and ClC-2, cAMP-dependent and voltage-activated chloride channels, respectively, are highly expressed in salivary gland duct cells. It is possible that activation of these channels by cAMP may result in fluid secretion. However, IPR-stimulated secretion was unaffected in mice lacking functional Cftr (CftrF508/F508) or ClC-2 (Clcn2-/-) Cl- channels. Beta-adrenergic stimulation causes cell swelling: The time course for activation of IPR-stimulated fluid secretion closely correlated with the time course for the IPR-induced increase in acinar cell volume suggesting that swelling may have activated a volume-sensitive Cl- channel (VRAC). Indeed, the non-selective Cl- channel blocker NPPB abolished fluid secretion, consistent with a Cl- channel playing an important role in this IPR-stimulated process. Ae4 (Slc4a9) Anion Exchangers Drive Cl- Uptake-dependent Fluid Secretion: We found that both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion. Moreover, our results strongly suggest that Ae4 is an electroneutral Cl(-)/nonselective cation-HCO3 (-) exchanger. We postulate that the physiological role of Ae4 in secretory cells is to promote Cl(-) influx in exchange for K(+)(Na(+)) and HCO3 (-) ions. In summary, we demonstrate that Tmem16A Cl- channels are essential for muscarinic, Ca2+-dependent fluid secretion by the adult mouse salivary gland. In contrast, beta-adrenergic-induced fluid secretion was independent of Tmem16A, Cftr and ClC-2 Cl- channels. The acinar cell swelling observed during IPR stimulation strongly suggests the involvement of the NPPB-sensitive, volume-regulated anion channel in this novel IPR-stimulated fluid secretion pathway in salivary glands.